CN108220233B - Cell separation instrument surface treatment method, related instrument, and method for rapidly and efficiently separating peripheral blood rare cells or circulating tumor cells - Google Patents
Cell separation instrument surface treatment method, related instrument, and method for rapidly and efficiently separating peripheral blood rare cells or circulating tumor cells Download PDFInfo
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Abstract
The invention provides a method for quickly and efficiently separating peripheral blood rare cells, which comprises the following steps: (1) separating white blood cells in peripheral blood, and resuspending floating white blood cells by using a cell diluent to obtain a white blood cell solution; (2) mixing and incubating the magnetic sphere-leukocyte surface biomarker antibody with a leukocyte solution to obtain an incubation solution; (3) placing a part of a circular pipeline with the inner wall closed by fibrinogen in a magnetic field, diluting an incubation solution by using a cell diluent to obtain an incubation diluent, enabling the incubation diluent to circularly flow in the circular pipeline, adsorbing a magnetic ball in the incubation diluent by using the magnetic field, and collecting liquid components in the circular pipeline. Also provides a method for rapidly and efficiently separating peripheral blood circulation tumor cells, a surface treatment method for a cell separation device and a related device. The method for separating the peripheral blood rare cells or the circulating tumor cells can quickly and efficiently separate the peripheral blood rare cells or the circulating tumor cells, is ingenious in design, simple and convenient to operate, and is suitable for large-scale popularization and application.
Description
Technical Field
The invention relates to the technical field of cell separation, in particular to the technical field of separation of peripheral blood rare cells or circulating tumor cells, and specifically relates to a surface treatment method of a cell separation device, a related device, and a rapid and efficient separation method of peripheral blood rare cells or circulating tumor cells.
Background
The detection of circulating tumor cells in peripheral blood has great clinical diagnosis value as an important content of tumor liquid biopsy. The major difficulties faced in the detection of circulating tumor cells in peripheral blood include: 1. the number of circulating tumor cells is very small, and only a few to tens of circulating tumor cells exist in a 7.5ml blood sample; 2. the proportion of the sample is extremely low, and a plurality of or tens of cells are distributed in 5 to 7 million normal cells; 3. in the absence of clear cell surface markers of circulating tumor cells, the cell surface marker molecule of circulating tumor cells which is widely used at present is EpCAM, but more and more researches show that a considerable number of circulating tumor cells express low or no EpCAM molecules; 4. lack of a physical property that clearly distinguishes normal cells. Some methods for enriching and separating circulating tumor cells according to cell density or cell size have the defects of complicated and time-consuming treatment and lack of characteristics.
The current research direction tends to adopt a negative enrichment method, namely, normal cells in a peripheral blood sample are removed to obtain peripheral blood rare cells, and circulating tumor cells are reserved to the maximum extent. Normal cells in peripheral blood all express CD45 cell surface molecules, so the method widely adopted at present is to modify the normal cells by using magnetic spheres coated with specific CD45 antibodies, and then carry out rapid separation under the action of a magnetic field. However, the expected separation effect is often not obtained by this method, for reasons including: 1. the surface of the device contacting with the cells in the cell treatment process can nonspecifically adsorb the cells, so that 10% -60% of target cells are lost, and the loss cannot be effectively improved after general surface treatment such as treatment by serum with certain concentration is carried out; 2. when the magnetic field is applied for separation, the acting distance of the magnetic field is short, so that the modification of the magnetic spheres on the cells can be generally carried out in a small reaction system, generally less than 1-5ml, which causes the density of the cells in the sample to be overlarge, reaching 1 million-7 million per ml, and in the process of magnetic field application separation, the cells modified by the magnetic spheres can be separated together with a large amount of cells modified by non-magnetic spheres, so that the target cells are greatly lost.
Some methods using microfluidic chips perturb the flow path of the cells by the characteristics of the microfluid to enhance the contact between the cell surface and the antibody modified on the inner wall of the flow channel, thereby improving the capture efficiency of the target cells; another microfluid chip adopts the characteristics of microfluid or the characteristics of acoustic wave acting on fluid to separate the cells with different particle sizes in the fluid in a layering way; in any microfluidic chip, the speed of processing the sample by the microfluid is slow, and the time for processing 1ml of sample is generally more than 2-3 hours.
Therefore, it is required to provide a method for rapidly and efficiently separating peripheral blood rare cells or circulating tumor cells, which can rapidly and efficiently separate peripheral blood rare cells or circulating tumor cells.
Disclosure of Invention
In order to overcome the defects in the prior art, an object of the present invention is to provide a method for rapidly and efficiently separating rare cells from peripheral blood, which can rapidly and efficiently separate rare cells from peripheral blood and is suitable for large-scale popularization and application.
The invention also aims to provide a method for quickly and efficiently separating rare cells in peripheral blood, which is ingenious in design, simple and convenient to operate and suitable for large-scale popularization and application.
Another objective of the present invention is to provide a method for rapidly and efficiently separating peripheral blood circulating tumor cells, which is capable of rapidly and efficiently separating peripheral blood circulating tumor cells and is suitable for large-scale popularization and application.
The invention also aims to provide a method for quickly and efficiently separating peripheral blood circulation tumor cells, which has the advantages of ingenious design and simple and convenient operation and is suitable for large-scale popularization and application.
The invention also aims to provide a surface treatment method for the cell separation device, and the cell separation device adopting the surface treatment method can effectively reduce the nonspecific adsorption of cells in the cell operation process, thereby reducing the loss of target cells, improving the enrichment and separation efficiency of the target cells and being suitable for large-scale popularization and application.
The invention also aims to provide a surface treatment method of the cell separation device, which has the advantages of ingenious design and simple and convenient operation and is suitable for large-scale popularization and application.
Another object of the present invention is to provide a cell separation device with surface treatment, which can effectively reduce the nonspecific adsorption of cells during the cell manipulation process, thereby reducing the loss of target cells, improving the enrichment and separation efficiency of target cells, and being suitable for large-scale popularization and application.
Another objective of the present invention is to provide a cell separation device with surface treatment, which has smart design and simple structure, and is suitable for large-scale popularization and application.
In order to achieve the above object, according to a first aspect of the present invention, there is provided a method for treating a surface of a cell separation instrument, characterized by sealing a cell-contacting surface of the cell separation instrument with fibrinogen.
Preferably, the fibrinogen is in the form of a fibrinogen solution, and the sealing treatment is performed by infiltrating the fibrinogen solution on the cell-contacting surface of the cell separation device.
More preferably, the fibrinogen solution contains fibrinogen that is human fibrinogen or bovine fibrinogen.
More preferably, the fibrinogen solution is 0.1% (w/v) to 60% (w/v) fibrinogen solution.
Preferably, the time of the blocking treatment is 15 minutes to 60 minutes.
Preferably, the cell separation instrument is a liquid holding vessel, a liquid flow-through line or a pipette tip.
More preferably, the inner diameter of the liquid flow pipe is 2mm to 15mm, and the outer diameter of the liquid flow pipe is not more than 20 mm.
Further, the inner diameter of the liquid flowing pipeline is 5mm-10 mm.
In a second aspect of the present invention, there is provided a surface-treated cell separation device, wherein the surface-treated cell separation device has fibrinogen enclosed on the cell-contacting surface.
In a third aspect of the present invention, there is provided a cell separation device having a surface treated by the above-mentioned surface treatment method for a cell separation device.
In a fourth aspect of the present invention, a method for rapidly and efficiently separating rare cells from peripheral blood is provided, which is characterized by comprising the following steps:
(1) separating white blood cells in peripheral blood, and resuspending the white blood cells by using a cell diluent to obtain a white blood cell solution;
(2) mixing and incubating a magnetic sphere-leukocyte surface biomarker antibody with the leukocyte solution to obtain an incubation solution;
(3) placing a part of a circular pipeline with the inner wall closed by fibrinogen in a magnetic field, diluting the incubation solution by using another cell diluent to obtain an incubation diluent, then enabling the incubation diluent to circularly flow in the circular pipeline, adsorbing the magnetic ball in the incubation diluent through the magnetic field, and collecting liquid components in the circular pipeline.
Preferably, in the step (1), the red blood cells and platelets in the peripheral blood are removed by density gradient centrifugation or red blood cell lysis.
Preferably, in step (2), the leukocyte surface biomarker antibody is a CD45 antibody.
Preferably, in the step (3), the inner diameter of the annular pipeline is 2mm-15mm, and the outer diameter of the annular pipeline is not more than 20 mm.
More preferably, in the step (3), the inner diameter of the annular pipeline is 5mm-10 mm. Preferably, in the step (3), a part of the annular pipeline is extruded into a flat pipeline before the circulating flow, and the distance between two opposite pipe walls with short distance of the flat pipeline is 1mm-10 mm.
Preferably, in the step (3), the circulation flow rate is 0.1ml to 10ml per minute.
More preferably, in the step (3), the circulation flow rate is 0.2ml to 5ml per minute.
Further, in the step (3), the circulation flow rate is 0.5ml to 1ml per minute.
Preferably, after the step (3), the method for rapidly and efficiently separating rare cells in peripheral blood further comprises the steps of: (4) maintaining a portion of said loop in said magnetic field, washing said loop with additional said cell dilution and collecting the wash solution.
In a fifth aspect of the present invention, a method for rapidly and efficiently separating peripheral blood circulating tumor cells is provided, which is characterized by comprising the following steps:
A. separating white blood cells in peripheral blood, and resuspending the white blood cells by using a cell diluent to obtain a white blood cell solution;
B. mixing and incubating the magnetic sphere-circulating tumor cell surface biomarker antibody with the leukocyte solution to obtain an incubation solution;
C. placing a part of a circular pipeline with the inner wall closed by fibrinogen in a magnetic field, diluting the incubation solution by using another cell diluent to obtain an incubation diluent, then enabling the incubation diluent to circularly flow in the circular pipeline, adsorbing the magnetic ball in the incubation diluent through the magnetic field, discarding a liquid component in the circular pipeline, removing the magnetic field, cleaning the circular pipeline by using another cell diluent, and collecting a cleaning solution.
Preferably, in the step A, the red blood cells and platelets in the peripheral blood are removed by using a density gradient centrifugation or red blood cell lysis method.
Preferably, in step B, the circulating tumor cell surface biomarker antibody is an EpCAM antibody.
Preferably, in the step C, the inner diameter of the annular pipeline is 2mm-15mm, and the outer diameter of the annular pipeline is not more than 20 mm.
More preferably, in the step C, the inner diameter of the annular pipeline is 5mm-10 mm.
Preferably, in the step C, a part of the annular pipeline is extruded into a flat pipeline before the circulating flow, and the distance between two opposite pipe walls with short distance of the flat pipeline is 1mm-10 mm.
Preferably, in the step C, the circulation flow rate is 0.1ml-10ml per minute.
More preferably, in the step C, the circulation flow rate is 0.2ml to 5ml per minute.
Further, in the step (3), the circulation flow rate is 0.5ml to 1ml per minute.
Preferably, after the step C, the method for rapidly and efficiently separating the peripheral blood circulation tumor cells further comprises the steps of: D. flushing the loop with additional cell diluent and collecting the flushing liquid.
The invention has the beneficial effects that:
1. the cell separation device surface treatment method adopts the fibrinogen to seal and treat the contact cell surface of the cell separation device, and the cell separation device adopting the method to carry out surface treatment can effectively reduce the nonspecific adsorption of cells in the cell operation process, thereby reducing the loss of target cells, improving the enrichment and separation efficiency of the target cells and being suitable for large-scale popularization and application.
2. The surface treatment method of the cell separation device adopts the fibrinogen to seal the contact cell surface of the cell separation device, has smart design and simple and convenient operation, and is suitable for large-scale popularization and application.
3. The surface of the cell separation device for surface treatment, which is contacted with cells, is sealed with fibrinogen, and the surface treatment method of the cell separation device is adopted for treatment, so that the nonspecific adsorption of the cells in the cell operation process can be effectively reduced, the loss of target cells can be reduced, the enrichment and separation efficiency of the target cells can be improved, and the cell separation device is suitable for large-scale popularization and application.
4. The surface of the cell separation device for surface treatment, which is contacted with cells, is sealed with fibrinogen, and the surface treatment method for the cell separation device is adopted for treatment, so that the surface treatment device for surface treatment is ingenious in design, simple in structure and suitable for large-scale popularization and application.
5. The method for quickly and efficiently separating the rare cells in the peripheral blood comprises the following steps: (1) separating white blood cells in peripheral blood, and resuspending the white blood cells by adopting a cell diluent to obtain a white blood cell solution; (2) mixing and incubating the magnetic sphere-leukocyte surface biomarker antibody with a leukocyte solution to obtain an incubation solution; (3) the method comprises the steps of placing a part of a circular pipeline with the inner wall closed by fibrinogen in a magnetic field, diluting an incubation solution by adopting another cell diluent to obtain an incubation diluent, enabling the incubation diluent to circularly flow in the circular pipeline, adsorbing a magnetic ball in the incubation diluent through the magnetic field, and collecting liquid components in the circular pipeline, so that rare cells in peripheral blood can be rapidly and efficiently separated, and the method is suitable for large-scale popularization and application.
6. The method for quickly and efficiently separating the rare cells in the peripheral blood comprises the following steps: (1) separating white blood cells in peripheral blood, and resuspending the white blood cells by adopting a cell diluent to obtain a white blood cell solution; (2) mixing and incubating the magnetic sphere-leukocyte surface biomarker antibody with a leukocyte solution to obtain an incubation solution; (3) the method comprises the steps of placing a part of a circular pipeline with the inner wall closed by fibrinogen in a magnetic field, diluting an incubation solution by adopting another cell diluent to obtain an incubation diluent, enabling the incubation diluent to circularly flow in the circular pipeline, adsorbing a magnetic ball in the incubation diluent through the magnetic field, and collecting liquid components in the circular pipeline.
7. The method for quickly and efficiently separating the peripheral blood circulation tumor cells comprises the following steps: A. separating white blood cells in peripheral blood, and resuspending the white blood cells by adopting a cell diluent to obtain a white blood cell solution; B. mixing and incubating the magnetic sphere-circulating tumor cell surface biomarker antibody with a leukocyte solution to obtain an incubation solution; C. placing a part of a circular pipeline with the inner wall closed by fibrinogen in a magnetic field, diluting an incubation solution by adopting another cell diluent to obtain an incubation diluent, then enabling the incubation diluent to circularly flow in the circular pipeline, adsorbing a magnetic ball in the incubation diluent through the magnetic field, and discarding a liquid component in the circular pipeline. And removing the magnetic field, cleaning the annular pipeline by adopting additional cell diluent and collecting the cleaning solution, so that the peripheral blood circulating tumor cells can be quickly and efficiently separated, and the method is suitable for large-scale popularization and application.
8. The method for quickly and efficiently separating the peripheral blood circulation tumor cells comprises the following steps: A. separating white blood cells in peripheral blood, and resuspending the white blood cells by adopting a cell diluent to obtain a white blood cell solution; B. mixing and incubating the magnetic sphere-circulating tumor cell surface biomarker antibody with a leukocyte solution to obtain an incubation solution; C. placing a part of a circular pipeline with the inner wall closed by fibrinogen in a magnetic field, diluting an incubation solution by adopting another cell diluent to obtain an incubation diluent, then enabling the incubation diluent to circularly flow in the circular pipeline, adsorbing a magnetic ball in the incubation diluent through the magnetic field, and discarding a liquid component in the circular pipeline. And the magnetic field is removed, the annular pipeline is cleaned by adopting other cell diluent, and the cleaning fluid is collected, so that the design is ingenious, the operation is simple and convenient, and the method is suitable for large-scale popularization and application.
These and other objects, features and advantages of the present invention will become more fully apparent from the following detailed description and appended claims, and may be realized by means of the instrumentalities, devices and combinations particularly pointed out in the appended claims.
Detailed Description
The invention provides a cell separation device surface treatment method, which is used for reducing non-specific adsorption of cells on the cell contact surface of a device in the cell treatment process.
The fibrinogen may be any suitable fibrinogen, preferably the fibrinogen is human fibrinogen or bovine fibrinogen.
The fibrinogen may take any suitable form, and preferably, the fibrinogen is in the form of a fibrinogen solution which is allowed to infiltrate the cell-contacting surface of the cell separation device for the sealing treatment.
The fibrinogen solution may have any suitable concentration, and more preferably, the fibrinogen solution is from 0.1% (w/v) to 60% (w/v) fibrinogen solution.
The time of the blocking treatment can be determined according to needs, and preferably, the time of the blocking treatment is 15 minutes to 60 minutes.
The cell separation apparatus may be any suitable cell separation apparatus, and is any apparatus that comes into contact with cells during cell separation, and preferably, the cell separation apparatus is a liquid holding vessel, a liquid flow line, or a pipette tip.
The liquid flow conduit may be of suitable dimensions, more preferably the liquid flow conduit has an internal diameter of from 2mm to 15mm and an external diameter of no more than 20 mm. Further, the inner diameter of the liquid flowing pipeline is 5mm-10 mm.
The invention also provides a surface-treated cell separator, which is formed by sealing fibrinogen on the surface of a contact cell and can be treated by the surface treatment method of the cell separator.
By adopting the cell separation device with the surface treatment, the invention also provides a method for quickly and efficiently separating rare cells in peripheral blood, which comprises the following steps:
(1) separating white blood cells in peripheral blood, and resuspending the white blood cells by using a cell diluent to obtain a white blood cell solution;
(2) mixing and incubating a magnetic sphere-leukocyte surface biomarker antibody with the leukocyte solution to obtain an incubation solution;
(3) placing a part of a circular pipeline with the inner wall closed by fibrinogen in a magnetic field, diluting the incubation solution by using another cell diluent to obtain an incubation diluent, then enabling the incubation diluent to circularly flow in the circular pipeline, adsorbing the magnetic ball in the incubation diluent through the magnetic field, and collecting liquid components in the circular pipeline.
The annular conduit can be considered as the liquid flow conduit described above.
In the step (1), the red blood cells and platelets in the peripheral blood can be removed by any suitable means, and preferably, in the step (1), the red blood cells and platelets in the peripheral blood are removed by using a density gradient centrifugation method or a red blood cell lysis method.
In step (2), the antibody against the leukocyte surface biomarker may be any suitable antibody against the leukocyte surface biomarker, and preferably, in step (2), the antibody against the leukocyte surface biomarker is an antibody against CD 45.
In the step (3), the annular pipeline may have a suitable size, and preferably, in the step (3), the inner diameter of the annular pipeline is 2mm-15mm, and the outer diameter of the annular pipeline does not exceed 20 mm. More preferably, in the step (3), the inner diameter of the annular pipeline is 5mm-10 mm.
In order to separate peripheral blood rare cells better, in the step (3), a part of the annular pipeline is extruded into a flat pipeline before the circulating flow, and the distance between two opposite pipe walls with short distance of the flat pipeline is 1mm-10 mm.
The speed of the circulating flow can be determined according to the requirement, and preferably, in the step (3), the speed of the circulating flow is 0.1ml-10ml per minute. More preferably, in the step (3), the circulation flow rate is 0.2ml to 5ml per minute. Further, in the step (3), the circulation flow rate is 0.5ml to 1ml per minute.
In order to be able to separate the rare cells in the peripheral blood more fully, preferably, after the step (3), the method for rapidly and efficiently separating the rare cells in the peripheral blood further comprises the steps of: (4) maintaining a portion of said loop in said magnetic field, washing said loop with additional said cell dilution and collecting the wash solution.
By adopting the cell separation device with the surface treatment, the invention also provides a method for quickly and efficiently separating peripheral blood circulation tumor cells, which comprises the following steps:
A. separating white blood cells in peripheral blood, and resuspending the white blood cells by using a cell diluent to obtain a white blood cell solution;
B. mixing and incubating the magnetic sphere-circulating tumor cell surface biomarker antibody with the leukocyte solution to obtain an incubation solution;
C. placing a part of a circular pipeline with the inner wall closed by fibrinogen in a magnetic field, diluting the incubation solution by using another cell diluent to obtain an incubation diluent, then enabling the incubation diluent to circularly flow in the circular pipeline, adsorbing the magnetic ball in the incubation diluent through the magnetic field, discarding a liquid component in the circular pipeline, removing the magnetic field, cleaning the circular pipeline by using another cell diluent, and collecting a cleaning solution.
The annular conduit can be considered as the liquid flow conduit described above.
In step a, the red blood cells and platelets in the peripheral blood can be removed by any suitable means, and preferably, in step a, the red blood cells and platelets in the peripheral blood are removed by using density gradient centrifugation or red blood cell lysis method.
In step B, the circulating tumor cell surface biomarker antibody may be any suitable circulating tumor cell surface biomarker antibody, preferably, in step B, the circulating tumor cell surface biomarker antibody is an EpCAM antibody.
In step C, the ring line may have a suitable size, and preferably, in step C, the inner diameter of the ring line is 2mm to 15mm, and the outer diameter of the ring line is not more than 20 mm. More preferably, in the step C, the inner diameter of the annular pipeline is 5mm-10 mm.
In order to better separate the peripheral blood circulation tumor cells, preferably, in the step C, a part of the annular pipeline is extruded into a flat pipeline before the circulation flow, and the distance between two opposite pipe walls with short distance of the flat pipeline is 1mm-10 mm.
The speed of the circulating flow can be determined according to the needs, and preferably, in the step C, the speed of the circulating flow is 0.1ml-10ml per minute. More preferably, in the step C, the circulation flow rate is 0.2ml to 5ml per minute. Further, in the step C, the circulation flow rate is 0.5ml to 1ml per minute.
In order to separate the peripheral blood circulating tumor cells more fully, preferably, after the step C, the method for rapidly and efficiently separating the peripheral blood circulating tumor cells further comprises the steps of: D. flushing the loop with additional cell diluent and collecting the flushing liquid.
The following related terms are referred to herein, with the following being expressly understood:
peripheral blood rare cells: it refers to cells in peripheral blood other than normal cells such as platelets, erythrocytes, leukocytes, etc., and includes circulating tumor cells.
Circulating tumor cells: the tumor cells actively or passively separated from primary or metastatic tumor focus sites and entering blood and lymph circulation are important detection objects of the current tumor liquid biopsy, such as MCF-7, SK-Br-3, sW60 and the like.
CD45 molecule: one cell surface protein molecule expressed on the surface of all normal leukocytes in peripheral blood is commonly used as a marker for normal cells.
Magnetic sphere-antibody: the method is characterized in that the antibody and the surface of the magnetic sphere are subjected to covalent cross-linking through a certain method, and the antibody and the surface of the magnetic sphere are used for modifying a target substance to be separated in a sample and can move and aggregate under the action of a magnetic field to realize separation from the sample.
DAPI (4,6-diamino-2-phenyl indole), a fluorescent dye that stains DNA molecules in the nucleus.
CD45-PE (CD 45-P-phycerythrin): an antibody molecule modified with a fluorescent molecule PE that specifically recognizes the CD45 molecule.
CK19-FITC (Cytokert 19-fluoroescein isothiocyanate) an antibody molecule specifically recognizing the CK19 antigen modified with the fluorescent dye molecule FITC.
And (3) sealing treatment, namely infiltrating the surface of the contact sample with certain protein or other organic components to reduce the nonspecific adsorption of the surface of the contact sample to the target in the sample.
And (3) forward selection: the method refers to that the target substance to be separated and enriched is directly separated from a mixture under the action of a magnetic field after being modified by a magnetic ball antibody.
And (3) negative selection: the method is characterized in that after a component similar to a separated and enriched target substance existing in a large amount in a solution is subjected to magnetic sphere antibody modification, the component is separated from the mixture under the action of a magnetic field, and the target substance is left in the mixture.
In order to clearly understand the technical contents of the present invention, the following examples are given in detail.
EXAMPLE 1 blocking of the vessel wall by fibrinogen
Sealing the surfaces of a centrifuge tube (made of PP) of 1.5ml and a centrifuge tube (made of PP) of 15ml and a silica gel hose (with the inner diameter of 5mm and the outer diameter of 9mm) of 30 cm in length by the following three methods, adding liquid containing a certain amount of lymphocytes and MCF cells into the centrifuge tube and the silica gel hose, and counting the cells in the liquid after 30 minutes of incubation reaction to calculate the yield of the cells. The three methods are 5% bovine fibrinogen (Shanghai leaf source, product number: 9001-32-5) solutions prepared by 1 and 10mMPBS (pH7.0) (SIGMA, Cat # P5493) respectively, and the surface is infiltrated for 30 minutes (hereinafter referred to as a first sealing method); 2. 5% BSA solution (hereinafter referred to as blocking method II) prepared from 10mM PBS (pH 7.0); 3. 30% bovine serum (Bioteck, Cat # P30-3302) prepared from 10mM PBS (pH7.0) (hereinafter referred to as blocking method III); 4. the control group adopts 10mPBS (pH7.0) solution (hereinafter referred to as a sealing method IV); 5. a 0.1% human fibrinogen (SIGMACAT # F3879) solution prepared from 10mMPBS (pH7.0) for 60 minutes (hereinafter referred to as the sealing method five); 6. a60% bovine fibrinogen (Shanghai leaf source, cat # 9001-32-5) solution prepared at 10mM PBS (pH7.0) was allowed to wet the surface for 15 minutes (hereinafter referred to as sealing method six). Table 1 lists the number of different cell types (lymphocytes (obtained by taking anticoagulated whole blood of healthy individuals and lysing erythrocytes) and human breast cancer cells MCF-7 (TCTU 74 as a cell bank product number in the institute of cell research, China academy of sciences)), added to different surfaces for different sealing methods, and finally the results and recovery rates are calculated by cell counting.
Table 1: surface sealing effect of fibrinogen
And (4) conclusion: according to the data in the table 1, the recovery rate of the three ways of the second, third and fourth sealing methods for the 1.5ml centrifuge tube and the 15ml centrifuge tube, whether the lymphocyte or the MCF-7 cell, is between 50% and 70%; the first sealing method, the fifth sealing method and the sixth sealing method can achieve the effect of 98 percent or more; for the silica gel hose, the recovery rates of the lymphocyte and the MCF-7 are between 50% and 70% by adopting the second, third and fourth sealing methods, and the recovery rates are high (more than 98%) without the first, fifth and sixth sealing methods. Therefore, the first sealing method, the fifth sealing method and the sixth sealing method have very good recovery rates no matter different sealing object surfaces or different cells, the recovery rate reaches 98% or more, the fibrinogen effects of the two sources used by the first sealing method, the fifth sealing method and the sixth sealing method have no obvious difference, and the nonspecific adsorption can be effectively eliminated. The surface closure is carried out by using the fibrinogen, so that the nonspecific adsorption of the contact cell surface to the cells can be effectively reduced, and the enrichment and separation efficiency of the target cells can be improved.
EXAMPLE 27.5 enrichment of circulating tumor cells in Whole blood samples (Positive selection)
1. Sample processing
Taking 10 healthy individual anticoagulated whole blood samples of 7.5ml, adding 100 rare cells such as MCF-7 tracer cells (TCTU 74 in cell bank of institute of Chinese academy of sciences), mixing, adding 8ml of erythrocyte lysate (RT 122-02 in Tiangen biotechnology limited company), standing at room temperature for 15 minutes, centrifuging at 300g for 20 minutes, collecting leukocytes, and repeating the above lysis steps;
2. sealing process
Sealing all the cell separation instruments in the following steps, including centrifuge tubes, liquid flow pipelines, liquid transfer suction heads and the like, on the cell contact surfaces by using a 5% bovine fibrinogen solution (the preparation method is the same as that of example 1) for 15-60 minutes;
3. separating and recovering
8 samples were taken as experimental groups and the following operations were performed: resuspending the obtained leukocytes with 300ul of cell dilution (SIGMA, Cat # P5493), adding 100ul of magnetosphere-antibody (EpCAM) (Meitianni Kaisha: 130061101) and mixing well with the cells, and incubating for 60 min in a mixer; diluting the incubated cells to a volume of 50ml with a cell diluent; taking a silicone tube with the length of 30 cm, the inner diameter of 5mm and the outer diameter of 9mm, fixing the silicone tube on a peristaltic pump, wherein one end of the silicone tube is connected to the lower end opening of a 50ml medical injector, and the other end of the silicone tube is placed into the upper end opening of the injector to form an annular pipeline; adding the sample into a syringe, and performing sample circulation flow at a flow rate of 2ml per minute under the driving of a peristaltic pump; placing a 4x40x90mm rectangular plate-shaped magnet above a section of pipe at the position of 10cm of the outlet of the peristaltic pump, and applying certain pressure to enable the inner cavity of the pipeline to be flat, wherein the height of the cavity is 1 mm; after 50 minutes, discarding the sample in the pipeline, removing the magnetic plate, washing the interior of the pipeline with 50ml of cell diluent under the condition of 5ml per minute of flow rate, and collecting cleaning fluid; then flushing the interior of the pipeline with 50ml of cell diluent under the condition of flow rate of 5ml per minute, and collecting cleaning fluid;
2 samples were taken as a control group and the following operations were performed: and (3) directly applying a magnetic field on the outer wall of the cell after the incubation is finished, discarding liquid components after the solution becomes clear, removing the magnetic field, resuspending the magnetic spheres by using cell diluent with the volume of 50ul (20-50ul can be used), and performing next step of flaking and staining detection.
4. Detection and results
After the obtained cell components are subjected to slide making, DAPI, CD45-PE and CK-19-FITC are adopted for fluorescent staining, an OlympusX83 full-automatic fluorescence scanning microscope is adopted for scanning and collecting images and analyzing, and DAPI positive-CK 19-FITC positive-CD 45 negative is taken to be judged as target cells. The results are shown in Table 2.
Table 2: forward enrichment and isolation of circulating tumor cells in 10 samples
This example was performed by adding a certain amount of labeled cells to 10 whole blood samples provided from normal healthy persons, wherein 8 experimental groups and 2 control groups were established. According to the detection result, the final MCF-7 detectable rate of the controls 1 and 2 is 20-40%, and the fibrinogen closed pipeline is used for collection, so that 100 tracer cells in 5 million white blood cells on average can be enriched within 50 minutes, and the recovery rate of more than 87% can be obtained.
EXAMPLE 37.5 enrichment isolation of rare cells in Whole blood samples (negative selection)
1. Sample processing
Taking 10 healthy individual anticoagulated whole blood samples of 7.5ml, adding 100 SK-Br-3 tracer cells (TCTU 225 in cell bank of institute of cell of Chinese academy of sciences) into each sample on average, mixing uniformly, adding 8ml of red blood cell lysate (RT 122-02 in the product number of Tiangen biotechnology limited), standing for 15 minutes at room temperature, centrifuging for 20 minutes at 300g, collecting white blood cells, and repeating the above lysis steps;
2. sealing process
The cell surfaces of all the cell separation instruments in the following steps including centrifuge tubes, liquid flow lines and pipette tips were sealed with 5% human fibrinogen solution (prepared as in example 1) for 15-60 minutes.
3. Separation and recovery
8 samples were selected as 8 cases of the experimental group, and the following operations were performed: the obtained leukocytes were resuspended in 300ul of cell diluent (Tiangen Biotech Co., Ltd.: RT122-02), and magnetic sphere-antibody (CD45) (Meitianni Kagaku Kogyo: 130045801) and the cells were added and mixed well and incubated in a mixer for 60 minutes; diluting the incubated cells to a volume of 50ml with a cell diluent; taking a silicone tube with the length of 30 cm, the inner diameter of 15mm and the outer diameter of 20mm, fixing the silicone tube on a peristaltic pump, wherein one end of the silicone tube is connected to a lower end opening of a 50ml medical injector, and the other end of the silicone tube is placed into an upper end opening of the injector; adding the sample into a syringe, and performing sample circulation flow at a flow rate of 2ml per minute under the driving of a peristaltic pump; placing a 4x40x90mm rectangular plate-shaped magnet above a section of pipe at the position of 10cm of the outlet of the peristaltic pump, and applying certain pressure to enable the inner cavity of the pipeline to be flat, wherein the height of the cavity is 10 mm; and after 50 minutes, putting one end of the outlet of the pipeline into a new collecting pipe, collecting nonmagnetic ball modified cell components, and after the sample in the pipeline flows out, cleaning the interior of the pipeline by using 50ml of cell dilution buffer solution under the condition of flow rate of 1.5ml per minute and collecting cleaning solution.
The remaining 2 samples served as control groups and were run as follows: and (3) directly applying a magnetic field to the cell after incubation on the outer wall of the tube, and transferring the liquid component to a new collecting tube after the solution becomes clear for further flaking and dyeing detection.
4. Detection and results
After the obtained cell components are subjected to slide making, DAPI, CD45-PE and CK-19-FITC are adopted for fluorescent staining, an OlympusX83 full-automatic fluorescence scanning microscope is adopted for scanning and collecting images and analyzing, and DAPI positive-CK 19-FITC positive-CD 45 negative is taken to be judged as target cells. The results are shown in Table 3.
Table 3: negative enrichment and separation of rare cells in 10 samples
This example was performed by adding a certain amount of labeled cells to 10 whole blood samples provided from normal healthy persons, wherein 8 experimental groups and 2 control groups were established. According to the detection result, the final detection rate of SK-Br-3 of the contrast 1 and the contrast 2 is 20-30%, and the fibrinogen closed pipeline is used for collection, 100 tracer cells in 5 million white blood cells on average can be enriched within 50 minutes, and the recovery rate of more than 90% is obtained, so that the capture efficiency of the tracer cells is obviously improved by the method.
Therefore, the invention provides a closed modification method of the cell contact surface of the cell separation device used in the enrichment and separation of rare cells in peripheral blood, which can effectively reduce the nonspecific adsorption of the cell contact surface of the cell separation device to the cells in the cell operation process, thereby improving the enrichment and separation efficiency of the rare cells; on the other hand, the method is characterized in that after the cells are modified by the magnetic ball-antibody, the sample circularly flows through the magnetic field at a certain concentration and flow rate, so that the rapid specific enrichment and separation of the cells of the high-throughput sample are realized.
Because the absolute number and proportion of rare cells in peripheral blood are extremely low, peripheral blood samples to be processed are generally 5ml to 15ml, generally 7.5ml, the number of white blood cells contained in the samples is 3 to 7 million, the number of red blood cells is 3 to 4 hundred million, and the number of platelets is 10 to 30 hundred million, while the number of rare cells to be separated and enriched (the circulating tumor cells are contained in the samples) is extremely low, the number of the circulating tumor cells is one to tens of cells, and the samples lack specific surface markers and physical properties. In order to effectively enrich and separate these rare cells, it is first necessary to remove red blood cells and platelets by density gradient centrifugation or red blood cell lysis to obtain leukocytes. The surface of normal leucocyte expresses CD45 molecule, so it can be modified by magnetic ball-monoclonal antibody which can be specifically identified and combined with the molecule, then removed under the action of magnetic field, to realize the enrichment and separation of rare cell. In the incubation reaction of the magnetic ball-antibody and the leucocyte and the subsequent separation process, if no blocking treatment is carried out, the surfaces of the used tube and vessel can nonspecifically adsorb the cells, so that a considerable amount of cells are lost, and simultaneously, the loss of rare cells with extremely small amount is caused, the loss is generally 10-60%. The invention adopts 0.1-60% of human or bovine fibrinogen prepared in PBS buffer solution to modify the surface of the cell separation device contacting with cells (hereinafter referred to as surface modification), so that a layer of fibrinogen is attached to the surface, thus effectively reducing nonspecific adsorption to the cells and reducing nonspecific adsorption to the cells to 2% or less, and no document reports such a method at present.
The cells modified by the magnetic ball-antibody usually make directional movement under the action of an external magnetic field, and the cells with the magnetic balls adsorbed on the surfaces gather to one side of the tube wall under the pushing or pulling of the magnetic balls, so that the separation from the solution is realized. Due to the strength of the external magnetic field and the characteristic that the magnetic field exponentially weakens with the increase of the distance, the distance acted by the magnetic field is generally not more than 10mm, so that the processing volume of the sample cannot be too large and is generally less than 1ml to be suitable for processing, and at the volume, the cell density is too large, which directly causes the obstruction or carrying effect in the movement of the magnetic bead modified cells. The invention provides a method for solving the contradiction: firstly, diluting a sample modified by the magnetic bead-antibody into a certain buffer solution, namely a cell diluent, wherein the commonly used buffer solution comprises 0.9 weight percent of physiological saline, 10mMPBS (pH7.0-7.5) and the like, the diluted volume can be 10-200ml, usually 25ml-50ml is the optimal volume, and the inner surface of the wall of a tube to be diluted must be subjected to surface modification; circulating the diluted sample by using a common adjustable liquid flow driving device, such as a flow adjustable peristaltic pump and a flexible plastic hose with a certain length and elasticity, wherein the inner wall of a flowing pipeline is required to be subjected to surface modification, the inner diameter of the pipeline is in a range of 2mm-15mm, 5mm-10mm is optimal, the outer diameter of the pipeline is not more than 20mm, the flowing speed is in a range of 0.1ml-10ml per minute, 0.2ml-5ml is more appropriate, and 0.5ml-1ml is optimal per minute; in the flowing process of a sample, the magnet is arranged above the pipeline, or the magnet can be extruded from top to bottom in a certain mode but cannot seal the inner cavity of the pipeline, and under the condition of ensuring the liquid to flow, the distance between the upper pipe wall and the lower pipe wall of the inner cavity is more than 1mm but less than 10 mm; when the sample flows through a sample volume equal to or greater than 2 times the volume of the sample, i.e., at least two sample volumes are circulated, the nonmagnetic sphere-antibody modified cell sample can be collected through the channel opening, and the channel can be washed with a volume of the same cell dilution (e.g., the same volume of the sample) at a flow rate equal to or less than the circulation flow rate; after the magnetic field is removed, the pipeline can be flushed by the same cell diluent with the volume not smaller than the volume in the pipeline at the flow rate not smaller than the circulating flow, meanwhile, the cell sample enriched by the magnetic beads, namely the magnetic ball-antibody modified cell sample, is collected, and the flushing can be repeated once.
In conclusion, the method for rapidly and efficiently separating the peripheral blood rare cells or the circulating tumor cells can rapidly and efficiently separate the peripheral blood rare cells or the circulating tumor cells, is ingenious in design, simple and convenient to operate, and suitable for large-scale popularization and application. The cell separation device surface treatment method can enable the cell separation device to effectively reduce the nonspecific adsorption of cells in the cell operation process, thereby reducing the loss of target cells, improving the enrichment and separation efficiency of the target cells, and being suitable for large-scale popularization and application.
It will thus be seen that the objects of the invention have been fully and effectively accomplished. The functional and structural principles of the present invention have been shown and described in the embodiments, and the embodiments may be modified without departing from the principles. Therefore, this invention includes all modifications encompassed within the spirit and scope of the claims.
Claims (25)
1. A cell separation device surface treatment method is characterized in that a contact cell surface of a cell separation device is subjected to sealing treatment by adopting fibrinogen, wherein the fibrinogen is human fibrinogen or bovine fibrinogen, the fibrinogen is in the form of a fibrinogen solution, the contacting cell surface of the cell separation device is infiltrated by the fibrinogen solution for sealing treatment, the fibrinogen solution is 0.1% (w/v) -60% (w/v) of the fibrinogen solution, the sealing treatment time is 15 minutes-60 minutes, the surface material of the cell separation device is PP or silica gel, and the cell separation device is used for separating lymphocytes or peripheral blood rare cells.
2. The method for surface treatment of a cell separation instrument according to claim 1, wherein the cell separation instrument is a liquid container, a liquid flow line or a pipette tip.
3. The method for surface treatment of a cell separation instrument according to claim 2, wherein the inner diameter of the liquid flow line is 2mm to 15mm, and the outer diameter of the liquid flow line is not more than 20 mm.
4. The method for surface treatment of a cell separation instrument according to claim 3, wherein the inner diameter of the liquid flow line is 5mm to 10 mm.
5. A cell separation device having a surface treated by the surface treatment method for a cell separation device according to any one of claims 1 to 4.
6. A method for rapidly and efficiently separating rare cells in peripheral blood is characterized by comprising the following steps:
(1) separating white blood cells in peripheral blood, and resuspending the white blood cells by using a cell diluent to obtain a white blood cell solution;
(2) mixing and incubating a magnetic sphere-leukocyte surface biomarker antibody with the leukocyte solution to obtain an incubation solution;
(3) placing a part of a loop pipeline with an inner wall closed by fibrinogen in a magnetic field, diluting the incubation solution by using another cell diluent to obtain an incubation diluent, circulating the incubation diluent in the loop pipeline, adsorbing the magnetic spheres in the incubation diluent by the magnetic field, and collecting a liquid component in the loop pipeline, wherein the fibrinogen is human fibrinogen or bovine fibrinogen, the fibrinogen is in the form of a fibrinogen solution, the fibrinogen solution infiltrates a contact cell surface of a cell separation device for a closing treatment, the cell separation device is the loop pipeline, the fibrinogen solution is 0.1% (w/v) -60% (w/v) of the fibrinogen solution, and the closing treatment is performed for 15-60 minutes, the surface material of the annular pipeline is silica gel.
7. The method for rapidly and efficiently separating rare cells in peripheral blood according to claim 6, wherein in the step (1), red blood cells and platelets in the peripheral blood are removed by density gradient centrifugation or red blood cell lysis.
8. The method for rapidly and efficiently separating rare cells from peripheral blood according to claim 6, wherein in the step (2), the leukocyte surface biomarker antibody is a CD45 antibody.
9. The method for rapidly and efficiently separating rare cells in peripheral blood according to claim 6, wherein in the step (3), the inner diameter of the circular pipeline is 2mm to 15mm, and the outer diameter of the circular pipeline is not more than 20 mm.
10. The method for rapidly and efficiently separating rare cells in peripheral blood according to claim 9, wherein in the step (3), the inner diameter of the circular pipeline is 5mm to 10 mm.
11. The method for rapidly and efficiently separating rare cells in peripheral blood according to claim 6, wherein in the step (3), a part of the annular tube is compressed into a flat tube before the circulating flow, and the distance between two opposite tube walls of the flat tube, which are spaced at a short distance, is 1mm to 10 mm.
12. The method for rapidly and efficiently separating rare cells in peripheral blood according to claim 6, wherein in the step (3), the circulating flow rate is 0.1ml to 10ml per minute.
13. The method for rapidly and efficiently separating rare cells in peripheral blood according to claim 12, wherein in the step (3), the circulating flow rate is 0.2ml to 5ml per minute.
14. The method for rapidly and efficiently separating rare cells in peripheral blood according to claim 13, wherein in the step (3), the circulating flow rate is 0.5 ml/min to 1 ml/min.
15. The method for rapidly and efficiently separating rare cells from peripheral blood according to claim 6, wherein after the step (3), the method for rapidly and efficiently separating rare cells from peripheral blood further comprises the steps of:
(4) maintaining a portion of said loop in said magnetic field, washing said loop with additional said cell dilution and collecting the wash solution.
16. A method for quickly and efficiently separating peripheral blood circulating tumor cells is characterized by comprising the following steps:
A. separating white blood cells in peripheral blood, and resuspending the white blood cells by using a cell diluent to obtain a white blood cell solution;
B. mixing and incubating the magnetic sphere-circulating tumor cell surface biomarker antibody with the leukocyte solution to obtain an incubation solution;
C. placing a part of a loop pipeline with an inner wall closed by fibrinogen in a magnetic field, diluting the incubation solution with another cell diluent to obtain an incubation diluent, circulating the incubation diluent in the loop pipeline, adsorbing the magnetic spheres in the incubation diluent by the magnetic field, discarding a liquid component in the loop pipeline, removing the magnetic field, cleaning the loop pipeline with another cell diluent, and collecting a cleaning solution, wherein the fibrinogen is human fibrinogen or bovine fibrinogen, the fibrinogen is in the form of a fibrinogen solution, the fibrinogen solution is infiltrated into a contact cell surface of a cell separation device for sealing treatment, the cell separation device is the loop pipeline, and the fibrinogen solution is 0.1% (w/v) -60% (w/v) of the fibrinogen solution, the time of the sealing treatment is 15-60 minutes, and the surface material of the annular pipeline is silica gel.
17. The method for rapidly and efficiently separating peripheral blood circulating tumor cells according to claim 16, wherein in the step A, red blood cells and platelets in the peripheral blood are removed by using a density gradient centrifugation or red blood cell lysis method.
18. The method for rapidly and efficiently isolating circulating tumor cells in peripheral blood according to claim 16, wherein in the step B, the circulating tumor cell surface biomarker antibody is EpCAM antibody.
19. The method for rapidly and efficiently separating circulating tumor cells in peripheral blood according to claim 16, wherein in the step C, the inner diameter of the circular tube is 2mm to 15mm, and the outer diameter of the circular tube is not more than 20 mm.
20. The method for rapidly and efficiently separating circulating tumor cells in peripheral blood according to claim 19, wherein in the step C, the inner diameter of the circular pipeline is 5mm to 10 mm.
21. The method of claim 16, wherein in step C, a portion of the annular tube is compressed into a flat tube before the circulation, and the distance between two opposite tube walls of the flat tube is 1mm-10 mm.
22. The method for rapidly and efficiently separating circulating tumor cells in peripheral blood according to claim 16, wherein in the step C, the circulating flow rate is 0.1 ml/min to 10 ml/min.
23. The method for rapidly and efficiently separating circulating tumor cells in peripheral blood according to claim 22, wherein in the step C, the circulating flow rate is 0.2 ml/min to 5 ml/min.
24. The method for rapidly and efficiently separating circulating tumor cells in peripheral blood according to claim 23, wherein in the step C, the circulating flow rate is 0.5 ml/min to 1 ml/min.
25. The method for rapidly and efficiently separating circulating tumor cells in peripheral blood according to claim 16, wherein after the step C, the method for rapidly and efficiently separating circulating tumor cells in peripheral blood further comprises the steps of:
D. flushing the loop with additional cell diluent and collecting the flushing liquid.
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